The present invention is directed to chimeric genes that express in cotton cells, plants and seeds, and encode insecticides and fungicides having substantially the insect toxicity and fungal toxicity of barley, nettle, and hevein lectins.
Chitin-binding proteins (lectins) are present in a wide range of plant species, including both monocots and dicots, even though these plants contain no chitin. They are believed to be defense-related, and many exhibit insecticidal and/or anti-fungal activities (Murdock et. al., 1990; Lerner, D. R. and Raikhel, N. V., 1992). Lectins exhibit specific carbohydrate-binding properties. Lectins are presumably defense-related proteins in plants that exert their effect by binding to N-acetylglucosamine in susceptible pest species (Schroeder, M. R. and Raikhel, N. V. 1992).
In purified form, barley, nettle, and hevein lectins have shown insecticidal and fungicidal activity against certain species of pests which are known to attack cotton (for example, Heliothis and Fusarium). Various methods are available to utilize lectins to control such pests, but all require providing these proteins sufficiently pure and in sufficient quantity to effect control of the target insect or pathogen. Even when available in sufficient purity or quantity, they must be applied to the crop in such a way so as to effectively reach the target species. Furthermore, because they are proteins, if topically applied to crops they are subject to light and protease inactivation before they can exert their controlling effect. Root associated pathogens are not readily treated with such preparations. Hence, lectins have not been available for use in controlling many serious pests of cotton, even though they might be effective were they available in pure enough and high enough concentrations.
By taking advantage of genetic engineering, a gene responsible for the production of a useful polypeptide can be transferred from a donor cell, in which the gene naturally occurs, to a host cell, in which the gene does not naturally occur; Cohen and Boyer, U.S. Pat. Nos. 4,237,224 and 4,468,464. There are, in fact, few inherent limits to such transfers. Genes can be transferred between viruses, bacteria, plants, and animals. In some cases, the transferred gene is functional, or can be made to be functional, in the host cell. When the host cell is a plant cell, whole plants can sometimes be regenerated from the cell.
Genes typically contain regions of DNA sequences including a promoter and a transcribed region. The transcribed region normally contains a 5xe2x80x2 untranslated region, a coding sequence, and a 3xe2x80x2 untranslated region.
The promoter contains the DNA sequence necessary for the initiation of transcription, during which the transcribed region is converted into mRNA. In eukaryotic cells, the promoter is believed to include a region recognized by RNA polymerase and a region which positions the RNA polymerase on the DNA for the initiation of transcription. This latter region, which is referred to as the TATA box, usually occurs about 30 nucleotides upstream from the site of transcription initiation.
Following the promoter region is a sequence that is transcribed into mRNA but is not translated into polypeptide. This sequence constitutes the so-called 5xe2x80x2 untranslated region and is believed to contain sequences that are responsible for the initiation of translation, such as a ribosome binding site.
The coding region is the sequence that is just downstream from the 5xe2x80x2 untranslated region in the DNA or the corresponding RNA. It is the coding region that is translated into polypeptides in accordance with the genetic code. Bacillus thuringiensis, for example, has a gene with a coding sequence that translates into the amino acid sequence of an insecticidal crystal protein.
The coding region is followed by a sequence that is transcribed into mRNA, but is not translated into polypeptide. This sequence is called the 3xe2x80x2 untranslated region and is believed to contain a signal that leads to the termination of transcription and, in eukaryotic mRNA, a signal that causes polyadenylation of the transcribed mRNA strand. Polyadenylation of the mRNA is believed to have processing and transportation functions.
Natural genes can be transferred in their entirety from a donor cell to a host cell. It is often preferable, however, to construct a gene containing the desired coding region with a promoter and, optionally, 5xe2x80x2 and 3xe2x80x2 untranslated regions that do not, in nature, exist in the same gene as the coding region. Such constructs are known as chimeric genes.
Barley lectin is a vacuolar protein synthesized with an amino-terminal signal sequence for entering the secretory pathway and a carboxyl-terminal propeptide necessary for proper targeting to the vacuole (Bednarek, S. Y., and Raikhel, N. V., 1991). The glycosylated carboxyl-terminal propeptide (CTPP) is removed before or concomitant with the deposition of the mature, active protein in vacuoles (Bednarek, et al., 1990). Mature barley lectin is a dimeric protein composed of two identical 18-kilodalton polypeptides (Wilkins, T. A., Bednarek, S. Y. and Raikhel, S. V., 1990). The nucleotide sequence and deduced amino acid sequence of the barley lectin coding region (barley lectin cDNA clone BLc3) has been reported (see Lemer and Raikhel, 1989; and U.S. Pat. No. 5,276,269, incorporated herein by reference). A chimeric gene construct was created by fusing the BLc3 coding region to the CaMV 35S promoter, and transferring the chimeric gene construct into tobacco plants via Agrobacterium tumefaciens mediated transformation (U.S. Pat. No. 5,276,269). Plants were reported to exhibit insecticidal and fungicidal properties.
A full length cDNA clone (HEV1) encoding Hevea brasiliensis lectin was isolated from a H. brasiliensis latex cDNA library, sequenced, and characterized (see Broekaert et al., 1990; Lee et al., 1991; and U.S. Pat. No. 5,187,262, incorporated herein by reference). Briefly, HEV1 is 1018 nucleotides long and includes an open reading frame of 204 amino acids. The deduced amino acid sequence contains a putative signal sequence of 17 amino acid residues followed by a 187 amino acid polypeptide. The amino-terminal region of 43 amino acids is identical to hevein and shows homology to several chitin-binding proteins and to the amino-termini of wound-induced genes in potato and poplar. Northern blots, using HEV1 cDNA as a probe, showed that the gene is induced by wounding and the plant hormones abscisic acid and ethylene. Accumulation of these transcripts was seen in leaves, stems, and latex, but not in roots. Chimeric gene constructs fusing the hevein coding region with heterologous promoters were not reported. However, tests with hevein protein showed antifungal activity against Trichoderma, Phycomyces, Botrytis, Septoria, Pyricularia, and Fusarium. The observed activities differed from those of wheat germ aglutinin (another lectin). Furthermore, hevein anti-fungal activity was found to be stable even after heating to 90xc2x0 C., a condition under which certain chitinase activities are completely destroyed.
A full length cDNA encoding the nettle lectin (Urtica dioica agglutinin) has been cloned, sequenced, and characterized (Lerner and Raikhel, 1992). The protein is made up of 374 amino acids. 21 are a putative signal sequence and 86 amino acids encode the two chitin-binding domains of nettle lectin. These are fused to a 19 amino acid xe2x80x9cspacerxe2x80x9d domain and a 244 amino acid carboxyl extension with partial identity to a chitinase catalytic domain.
This gene represents another lectin heretofore unavailable as a source for resistance to important cotton insect and fungal pathogens.
The studies noted above underscore the complexity of the biochemistry of plant lectins. These are proteins which must be processed properly and transported into the proper subcellular compartment, usually a vacuole, where they are stored. In order to make use of these proteins in combating cotton pests, one viable approach is to generate chimeric gene constructs using various lectin genes and then transfer these into cotton using available transformation systems (see for example, Rangan et al., U.S. Pat. No. 5,244,802). Achieving an effective level of expression is not a given in heterologous systems. There would be no guarantee that the proteins would not have some unexpected toxic effect on the cotton plant itself, or that the proteins would exhibit the predicted pattern of activity. Furthermore, as noted above, some target pests attack plant tissues (for example, roots) in which some of these lectins are not normally expressed in the plants from which they come. Hence, a lectin which might have activity against a given pest in a feeding assay following topical application to plant tissue (see, for example, Cavalieri et. al., U.S. Pat. No. 5,407,454), may not exhibit that same activity when expressed in vivo.
Cavalieri et al. provides somewhat suggestive evidence that a broad range of plant lectins may provide a level of control against certain corn pests. Unfortunately, those studies were carried out using isolated lectin preparations for which essentially no biochemical characterization was provided. Some may even have been from commercial providers, where composition can vary from preparation to preparation. Hence, commercial providers include lot numbers with their products so that problems can be traced back on a lot by lot basis. Purity of the preparations was not discussed by Cavalieri, nor did they provide information on how they obtained their lectins or discuss the actual number of different lectins which may have been present in a given preparation. Any plant species may produce several different lectins, and protein preparations are readily contaminated with multiple protein species which may be present in trace amounts, but have a significant effect, positive or negative, on observed activity. Hence, the preparations tried may have actually been mixtures of lectins and even other proteins derived from the plants in question. No data were provided on the source of the lectin preparations used, on their purity, or hence on which of the lectin genes in a given plant the actual activity observed was based. Such preparations could have distinctly different insecticidal and fungicidal activities than a lectin provided in purified form from the expression in planta of a single lectin gene.
The best way to provide a protein in purified form, and therefore be certain of its activity against a given pest, is to isolate the gene and express the protein in an in vitro system. Since genes for most of the lectins cited in their study have still not been cloned as of this date, in vitro expression of single, purified lectins for analysis was not possible at the time Cavalieri et al. reported their data. Suggestive as their data is with respect to certain corn pests, Cavalieri et al. do not provide a single example of activity against a serious pest of cotton. Hence, their study is suggestive, but does not disclose a single lectin, in purified form, which one might use to control a significant pest of cotton.
Conversely, proteins which do not have activity in a feeding assay following topical application to plant tissues, may have activity when expressed in vivo. This could particularly be true in cotton, where plants normally express a compound called gossypol which is known to suppress feeding of certain insect pests. Thus, there could be synergistic effects between gossypol and lectins in such a way so as to enhance the insecticidal activity of a given lectin against important cotton pests. Alternatively, gossypol expression could suppress feeding just enough so that the target insect might never consume a potentially lethal amount of lectin. Hence, one could not know the insecticidal or fungicidal effect of a lectin gene transferred into cotton until such cotton cells, plants, and seeds were created.
Raikhel (U.S. Pat. No. 5,276,269) showed that a chimeric barley lectin gene under control of the CaMV 35S promoter could be transferred into tobacco plants to produce a single species lectin protein which was transported properly and thereby create a plant with new insecticidal and fungicidal properties. With the further availability of the hevein (Raikhel, U.S. Pat. No. 5,187,262) and nettle genes due to cloning (Lemer and Raikhel, 1992), it has now become possible to create cotton plants expressing in highly purified form each of these lectins and to test those cells, plants, and seeds for the presence of new insecticidal and fungicidal activities.
It is an object of the present invention to provide cotton cells, plants, and seeds expressing chimeric barley, nettle, and hevein lectin genes in amounts and under conditions which are sufficient to impart substantially the pesticidal properties such as insecticidal and fungicidal properties of barley, nettle, and hevein lectins to said cotton cells, plants, and seeds.
It is a further object of the present invention to provide a method for killing cotton insect pests and pathogens by feeding them cotton cells, plants, and seeds containing chimeric genes that express pesticidal (for example, insecticidal and fungicidal) amounts of a toxin having substantially the insect toxicities and fungal toxicities of barley, nettle, and hevein lectins.
It is an additional object of the present invention to provide the genes and other DNA segments within the cotton cells, plants, and seeds associated with the above methods.
These and other objects of the present invention have been achieved by providing chimeric genes capable of expressing in cotton cells, plants, and seeds a polypeptide having substantially the pesticidal toxicity (for example, the insect toxicity) and fungal toxicity of barley, nettle, and hevein lectins, in plant cells in culture and plant cells in living plants and seeds; as well as methods for producing a toxin having substantially the pesticidal properties (for example, the insect toxicity and fungal toxicity) of barley, hevein, and nettle lectins in cotton cells, plants, and seeds; and methods for killing cotton pests such as insects by feeding them cotton cells, plants, and seeds containing genes that express these toxins.